The invention relates to a method of growing a confluent layer of cells on a porous or semi-permeable substrate and to apparatus for practicing the method.
For investigating a number of directed processes taking place on confluent cell layers grown on a substrate it is necessary to determine these processes on the apical side of the cell layer as well as those on the basal side thereof. To this end, both sides of a confluent cell layer grown on a porous or semi-permeable substrate must be contacted with perfusion fluid in a double perfusion chamber.
In order to place a substrate having a confluent cell layer grown thereon in a double perfusion chamber, it is necessary to mount the substrate in a holding and tensioning device which then is placed in the double perfusion chamber together with the substrate and the confluent cell layer thereon, the latter having grown for a particular period of time by mitotic division of seed cells.
It has been known to grow confluent cell layers on a porous or semi-permeable substrate by first spreading the substrate on the bottom surface of a so-called Petri dish to cover with one side thereof a medium containing the nutrient required for growing the cells. Thereafter, the seed cells are sown on the substrate and spread to confluence by multiple divisions. However, it has not been possible to this date to grow a confluent cell layer in a Petri dish on one side of a substrate mounted in a holding and tensioning device, said substrate being covered by the medium on both sides. Neither has it been possible to place and tension in a holding and tensioning device a substrate after a confluent cell layer had been grown thereon according to the aforesaid method. In the placing and tensioning process, forces act on the cell layer which cause it to sustain damage so as to disrupt the confluence of the cell layer which is required for the subsequent testing.
It is absolutely necessary to mount the conventional, extremely thin substrates, which may be as thin as 10 to 100 microns, and the confluent cell layers thereon in holding and tensioning means so as to enable the substrate to assume a planar condition in the double perfusion chamber. For this reason, it has not been possible in the past to perform tests in vitro, i.e. outside plant, animal or human bodies in a chamber of this type so as to observe and study directed biological, physiological, biochemical and pharmacological processes on cell layers that form physiological boundaries where corresponding activities take place. The aforesaid processes are enzymatic in nature and involve the discharge of substances to the cell environment in the confluent cell layer, the reception of substances from the cell environment, and the movement of substances from the apical side of the cells through the cell layer to the basal side of the cell, and vice versa. In a double perfusion chamber suited for investigating processes of this kind, both sides of the substrate and of the confluent cell layer thereon must be contacted uniformly and efficiently so that the perfusion fluid flows from both chambers reaching the measurement side may be observed and investigated separately.